Human cytomegalovirus (CMV) is a beta herpesvirus that causes significant pathology in individuals who are immunosuppressed, e.g., organ transplant recipients or individuals with Acquired Immune Deficiency Syndrome. There have been several reports of protein kinases induced by or associated with CMV infection. Mar et al., J. Gen. Virol. 57:149-156 (1981); Michelson et al., Eur. J. Biochem. 149:393-399 (1985); Michelson et al., Virology 134:259-268 (1984); Roby et al., J. Virol. 59:714-727 (1986); Britt et al., J. Virol. 59:185 (1986). UL97 is a CMV gene which is homologous to protein kinases. Chee et al., J. Gen. Virol. 70:1151-1160 (1989). An apparent substrate for UL97 is Ganciclovir (GCV), a nucleoside analog that has been used to inhibit CMV replication. GCV must be phosphorylated in order to be active. The UL97 gene product has been shown to be responsible for phosphorylation of GCV in CMV-infected cells, as a mutation in UL97 renders infected cells GCV resistant (GCV.sup.r), and also results in decreased GCV phosphorylation. Baldanti et al., J. Virol. 69:796-800 (1995); Hanson et al., Antimicrob. Agents Chemother. 39:1204-1205 (1995); Lorain et al., J. Virol. 68:4427-2231 (1994); Sullivan et al., Nature 358:162-165 (1992). UL97, when expressed in heterologous systems, can induce GCV phosphorylation in the absence of other CMV proteins. Littler et al., Nature 358:160-162 (1992); Metzger et al., J. Virol. 68:8423-8427 (1994). Extracts of E. coli expressing UL97 can phosphorylate GCV, and antiserum to a UL97 extract that has been partially purified from E. coli can immunoprecipitate GCV kinase activity from extracts of CMV-infected human cells. Littler et al., supra. Thus, it appears that UL97 mutations confer resistance to GCV by impairing the GCV kinase activity of UL97.
CMV UL97 is most closely related in sequence to a family of proteins encoded by all known herpesviruses. Chee et al., J. Gen. Virol. 70:1151-1160 (1989); deWind et al., J. Virol. 66:5200-5209 (1992); Smith et al., J. Virol. 63:450-455 (1989). HSV UL13, VZV ORF 47, and pseudorabies virus (PRV) UL13 are all associated with protein kinases. However, of these, only the PRV UL13 protein has been shown to be active when expressed in a heterologous system (de Wind et al., J. Virol. 64:4691-4696 (1990)); neither HSV UL13 nor VZV ORF 47 has exhibited activity following heterologous expression (Ng et al., Virology 191:9-18; Stevenson et al., J. Gen. Virol. 75:317-326 (1994)), and none of these proteins has yet been purified to show that it does not require any cellular proteins as cofactors.
Of the approximately twenty GCV.sup.r UL97 mutants that have been described, none appears to be a null mutant; i.e., no nonsense or frameshift mutations have been identified. Known UL97 mutations resulting in GCV resistance fall into two general classes. In one class, a segment of the UL97 protein that corresponds to a portion of the cAMP-dependent protein kinase that is involved in substrate specificity is affected. In the other class, the mutations map to a residue within a conserved region in UL97 that corresponds to the catalytic loop, but this particular residue is also implicated in substrate recognition. Thus, phosphorylation of GCV, which appears to be a fortuitous substrate of UL97, could be drastically impaired without major effects on phosphorylation of natural substrates of UL97. Accordingly, UL97 may be essential for CMV replication, and thus an important target for inhibition by antiviral drugs.
GCV has been widely used to treat CMV infection. However, GCV and other known anti-CMV drugs are limited by toxicity, pharmacokinetic problems, and/or lack of potency or efficacy in various settings. Moreover, GCV resistance in CMV-infected cells in vivo is a substantial problem. Biron, International Antiviral News 2:117-118 (1994); Drew et al., J. Infect. Dis. 163:716-719 (1991); Erice et al., New Engl. J. Med. 320:289-293 (1989); Tatarowicz et al., J. Infect. Dis. 166:904-907 (1992). It would therefore be desirable to develop screening assays for the identification of drugs that affect CMV infection and the pathology resulting therefrom.